Irene Romero

The immunomodulator PSK induces in vitro cytotoxic activity in tumour cell lines via arrest of cell cycle and induction of apoptosis

BackgroundProtein-bound polysaccharide (PSK) is derived from the CM-101 strain of the fungus Coriolus versicolor and has shown anticancer activity in vitro and in in vivo experimental models and human cancers. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the adjuvant treatment of gastric, esophageal, colorectal, breast and lung cancers. These studies have suggested the efficacy of PSK as an immunomodulator of biological responses. The precise molecular mechanisms responsible for its biological activity have yet to be fully elucidated.MethodsThe in vitro cytotoxic anti-tumour activity of PSK has been evaluated in various tumour cell lines derived from leukaemias, melanomas, fibrosarcomas and cervix, lung, pancreas and gastric cancers. Tumour cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of PSK on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in PSK-treated cells.ResultsPSK showed in vitro inhibition of tumour cell proliferation as measured by BrdU incorporation and viable cell count. The inhibition ranged from 22 to 84%. Inhibition mechanisms were identified as cell cycle arrest, with cell accumulation in G0/G1 phase and increase in apoptosis and caspase-3 expression. These results indicate that PSK has a direct cytotoxic activity in vitro, inhibiting tumour cell proliferation. In contrast, PSK shows a synergistic effect with IL-2 that increases PBL proliferation.ConclusionThese results indicate that PSK has cytotoxic activity in vitro on tumour cell lines. This new cytotoxic activity of PSK on tumour cells is independent of its previously described immunomodulatory activity on NK cells.

MHC class I molecules act as tumor suppressor genes regulating the cell cycle gene expression, invasion and intrinsic tumorigenicity of melanoma cells

The alteration of MHC class I (MHC-I) expression is a frequent event during cancer progression, allowing tumor cells to evade the immune system. We report that the loss of one major histocompatibility complex haplotype in human melanoma cells not only allowed them to evade immunosurveillance but also increased their intrinsic oncogenic potential. A second successive defect in MHC-I expression, MHC-I total downregulation, gave rise to melanoma cells that were more oncogenic per se in vivo and showed a higher proliferation rate and greater migratory and invasive potential in vitro. All these processes were reversed by restoring MHC-I expression via human leukocite antigen-A2 gene transfection. MHC-I cell surface expression was inversely correlated with intrinsic oncogenic potential. Modifications in the expression of various cell cycle genes were correlated with changes in MHC-I expression; the most important differences among the melanoma cell lines were in the transcriptional level of AP2-alpha, cyclin A1 and p21WAF1/CIP1. According to these results, altered MHC-I expression in malignant cells can directly increase their intrinsic oncogenic and invasive potential and modulate the expression of cell cycle genes. These findings suggest that human leukocite antigen class I molecules may act directly as tumor suppressor genes in melanoma.

The tumour suppressor Fhit positively regulates MHC class I expression on cancer cells

MHC class I (MHC‐I) molecules are ubiquitously expressed on the cells of an organism. Study of the regulation of these molecules in normal and disease conditions is important. In tumour cells, the expression of MHC‐I molecules is very frequently lost, allowing these cells to evade the immune response. Cancers of different histology have shown total loss of MHC‐I molecule expression, due to a coordinated transcriptional down‐regulation of various antigen‐processing machinery (APM) components and/or MHC‐I heavy chains. The mechanisms responsible for these alterations remain unclear. We determined the possible genes involved by comparing MHC‐I‐positive with MHC‐I‐negative murine metastases derived from the same fibrosarcoma tumour clone. MHC‐I‐negative metastases showed transcriptional down‐regulation of APM and MHC‐I heavy chains. The use of microarrays and subtraction cDNA libraries revealed four candidate genes responsible for this alteration, but two of them were ruled out by real‐time RT‐PCR analyses. The other two genes, AP‐2α and Fhit tumour suppressors, were studied by using siRNA to silence their expression in a MHC‐I‐positive metastatic cell line. AP‐2α inhibition did not modify transcriptional expression of APM components or MHC‐I heavy chains or surface expression of MHC‐I. In contrast, silencing of the Fhit gene produced the transcriptional down‐regulation of APM components and MHC‐I heavy chains and decreased MHC‐I surface expression. Moreover, transfection of Fhit in MHC‐I‐negative tumour cell lines restored MHC‐I cell surface expression. These data indicate that defects in Fhit expression may promote MHC‐I down‐regulation in cancer cells and allow escape from immunosurveillance#. Copyright

Metastases in Immune-Mediated Dormancy: A New Opportunity for Targeting Cancer

The aim of any anticancer treatment is to avoid, control, or eliminate disseminated tumor cells. Clinical and experimental evidence has revealed that metastases can remain in a latency state, that is, metastasis dormancy. Three mechanisms are thought to be involved in cancer dormancy: cellular dormancy, angiogenic dormancy, and immune-mediated dormancy. Here, we review the mechanisms and cells involved in immune-mediated cancer dormancy and discuss current and future immunotherapeutic strategies. Recent results indicate that the immune system can restrain disseminated cancer cells, promoting their permanent dormancy. CD8(+) T lymphocytes play a relevant role in maintaining immune equilibrium with metastatic dormant cells, and MHC class I surface expression on tumor cells may also be involved. Natural killer (NK) cells have an activator function that triggers a cytotoxic T lymphocyte (CTL) response. Furthermore, immune dormancy promotes cancer cell growth arrest and angiogenic control. Immunotherapeutic interventions in metastatic dormancy may help to control or eradicate cancer disease. Treatments that activate or increase the CTL immune response or reverse cancer cell-induced CTL immunosuppression might be useful to restrain or destroy metastatic cells. These objectives may be achieved by recovering or increasing MHC class I surface expression on cancer cells or even by activating NK cells. Immune-mediated metastasis dormancy provides an opportunity for targeting cancer in novel immune treatments.

T lymphocytes restrain spontaneous metastases in permanent dormancy

Tumor dormancy is a clinical phenomenon related to immune equilibrium during cancer immunoediting. The mechanisms involved in dormant metastases are poorly understood due to the lack of preclinical models. Here, we present a nontransgenic mouse model in which spontaneous metastases remain in permanent immunomediated dormancy with no additional antitumor treatment. After the injection of a GR9-B11 mouse fibrosarcoma clone into syngeneic BALB/c mice, all animals remained free of spontaneous metastases at the experimental endpoints (3-8 months) but also as long as 24 months after tumor cell injection. Strikingly, when tumor-bearing mice were immunodepleted of T lymphocytes or asialo GM1-positive cells, the restraint on dormant disseminated metastatic cells was relieved and lung metastases progressed. Immunostimulation was documented at both local and systemic levels, with results supporting the evidence that the immune system was able to restrain spontaneous metastases in permanent dormancy. Notably, the GR9-B11 tumor clone did not express MHC class I molecules on the cell surface, yet all metastases in immunodepleted mice were MHC class I-positive. This model system may be valuable for more in-depth analyses of metastatic dormancy, offering new opportunities for immunotherapeutic management of metastatic disease.

Immunotherapy eradicates metastases with reversible defects in MHC class I expression

Tumor or metastatic cells lose MHC class I (MHC-I) expression during cancer progression as an escape mechanism from immune surveillance. These defects in MHC-I may be reversible by cytokines or different agents (soft lesions) or irreversible due to structural defects (hard lesions). The nature of these MHC-I alterations might determine the success or failure of immunotherapy treatments. In this study, we have used an MHC-I-positive murine fibrosarcoma tumor clone, GR9-A7, which generates multiple lung and lymph node metastases with reversible MHC-I alterations after treatment with IFN-γ. Four different antitumor treatments were carried out after primary tumor excision to determine their capacity to inhibit spontaneous metastatic colonization of the GR9-A7 tumor clone. We found that 2 different immunotherapy protocols (CpG plus autologous irradiated-GR9-A7 cells and protein-bound polysaccharide K (PSK) and 1 chemoimmunotherapy (docetaxel plus PSK) induced eradication of metastases. In contrast, chemotherapy with docetaxel alone produced only partial reduction in the number of metastases. Flow cytometric analysis of lymphocyte populations showed an immunosuppression in GR9-A7 tumor-bearing host, which could be reverted by immunotherapy treatments. Our results suggest that irreversible or reversible MHC-I alterations in tumor target cells may determine its progression or regression independently of the type of immunotherapy used.

Class II transactivator-induced MHC class II expression in pancreatic cancer cells leads to tumor rejection and a specific antitumor memory response.

Objectives The loss of major histocompatibility complex (MHC) classes I and II is a well-known mechanism by which cancer cells are able to escape from immune recognition. In this study, we analyzed the expression of antigen processing and presenting molecules in 2 cell lines derived from mouse models of pancreatic ductal adenocarcinoma (PDA) and the effects of the re-expression of MHC class II on PDA rejection. Methods The PDA cell lines were analyzed for the expression of MHC class I, II, and antigen-processing molecules by flow cytometry or polymerase chain reaction. We generated stable PDA-MHC class II transactivator (CIITA) cells and injected them into syngeneic mice. The CD4 and CD8 T-cell role was analyzed in vitro and in vivo. Results Murine PDA cell lines were negative for MHC and antigen-processing molecules, but their expression was restored by exogenous interferon-&ggr;. CIITA-tumor cells were rejected in 80% to 100% of injected mice, which also developed long-lasting immune memory. In vitro assays and immunohistochemical analyses revealed the recruitment of T effector cells and CD8 T cells into the tumor area. Conclusions Overall, these data confirm that immunotherapy is a feasible therapeutic approach to recognize and target an aggressive cancer such as PDA.

MHC Intratumoral Heterogeneity May Predict Cancer Progression and Response to Immunotherapy

An individual tumor can present intratumoral phenotypic heterogeneity, containing tumor cells with different phenotypes that do not present irreversible genetic alterations. We have developed a mouse cancer model, named GR9, derived from a methylcholanthrene-induced fibrosarcoma that was adapted to tissue culture and cloned into different tumor cell lines. The clones showed diverse MHC-I phenotypes, ranging from highly positive to weakly positive MHC-I expression. These MHC-I alterations are due to reversible molecular mechanisms, because surface MHC-I could be recovered by IFN-γ treatment. Cell clones with high MHC-I expression demonstrated low local oncogenicity and high spontaneous metastatic capacity, whereas MHC-I-low clones showed high local oncogenicity and no spontaneous metastatic capacity. Although MHC-I-low clones did not metastasize, they produced MHC-I-positive dormant micrometastases controlled by the host immune system, i.e., in a state of immunodormancy. The metastatic capacity of each clone was directly correlated with the host T-cell subpopulations; thus, a strong decrease in cytotoxic and helper T lymphocytes was observed in mice with numerous metastases derived from MHC-I positive tumor clones but a strong increase was observed in those with dormant micrometastases. Immunotherapy was administered to the hosts after excision of the primary tumor, producing a recovery in their immune status and leading to the complete eradication of overt spontaneous metastases or their decrease. According to these findings, the combination of MHC-I surface expression in primary tumor and metastases with host T-cell subsets may be a decisive indicator of the clinical outcome and response to immunotherapy in metastatic disease, allowing the identification of responders to this approach.

MHC Class I Molecules and Cancer Progression: Lessons Learned from Preclinical Mouse Models

Major histocompatibility complex (MHC) class I molecules are expressed on the surface of nucleated cells and present peptides derived from endogenous proteins to CD8+ T-lymphocytes. In tumor cells, MHC class I (MHC-I) molecules may present peptides derived from tumor-associated antigens (TAAs), which are new proteins expressed or overexpressed in tumor cells. Presentation of these new peptides may allow recognition and destruction of tumor cells by CD8+ T-lymphocytes. Loss of MHC-I expression on tumor cells is a widespread and frequent mechanism developed to escape from immunosurveillance. Alteration in MHC-I in both human and murine experimental tumors has been widely reported. This chapter summarizes the role of MHC class I expression on cancer cells in tumor and metastatic progression, as well as its effect on the outcome of immunotherapy in murine experimental tumors. Results were obtained from different tumor clones derived from a fibrosarcoma induced by methylcholanthrene (MCA) in BALB/c mice, in addition to spontaneous metastases derived from these tumor clones, during more than 30 years of study on murine cancer model GR9. In this tumor model, results show an inverse correlation between MHC-I expression on tumor cells and primary tumor growth, i.e., MHC-I-negative tumors grew more rapidly compared to MHC-I-positive tumors. In contrast, a direct correlation was found between MHC-I expression on primary tumors and spontaneous metastatic capacity. Immunotherapy as an antimetastatic treatment was completely effective against MHC-I highly positive tumors and was partially effective on tumors with an intermediate level of MHC-I expression.

A novel preclinical murine model of immune-mediated metastatic dormancy

The mechanisms underlying cancer dormancy are poorly understood. We have developed a preclinical murine model in which immunosurveillance restrains spontaneous metastases in permanent dormancy. The model faithfully recapitulates human metastatic dormancy and may be useful to decipher the immune mechanisms constraining disease progression, thereby facilitating the development of novel immunotherapeutic approaches to control metastatic disease.